WO1993009838A1

WO1993009838A1 - Apparatus and method for long term electrical stimulation of heart muscle cells

Info

Publication number: WO1993009838A1
Application number: PCT/US1992/009605
Authority: WO
Inventors: Hans-Jorg Berger; John Lehr
Original assignee: Brigham And Women's Hospital
Priority date: 1991-11-15
Filing date: 1992-11-12
Publication date: 1993-05-27
Also published as: AU3069092A

Abstract

The present invention relates to an apparatus and a method for uniformly stimulating a large number of heart muscle cells, so that they will contract or beat, over long periods of time. The device uses an amplifier (320), connected to graphite electrodes (130), to produce a homogeneous electric field suitable for long term stimulation of the cells.

Description

TITLE OF THE INVENTION

APPARATUS AND METHOD FOR LONG TERM ELECTRICAL STIMULATION OF HEART MUSCLE CELLS

FIELD OF THE INVENTION

The present invention relates to apparatus and a method for stimulation of cells. More particularly, the device of the present invention uniformly stimulates a large number of heart muscle cells, so that they will contract or beat, over long periods of time as required for biochemical and molecular biological assays of these cells.

BACKGROUND OF THE INVENTION

Methods for culturing adult and neonatal heart cells (muscle and non-muscle) are well known, and the cultures are used for a variety of research and testing purposes. However, adult heart myocytes (muscle cells) are normally, quiescent, that is, they do not "beat" unless subjected to external stimulation.

Apparatus for small scale (i.e., small number of cells), short-term (i.e., approximately one hour) stimulation of heart muscle cells was described in the article by Karl erdan and Erland Erdmann, Preparation and Culture of Embryonic and Neonatal Heart Muscle Cells: Modification of Transport Activity, Methods in Enzymology, Volume 173, 1989. The article describes the methods for preparation and cultivation of cardiac muscle and non-muscle cells from chicken embryos and neonatal rats. The objective of the experiments was to study the active Na+ and K+ transport in the heart under various physiological and pathological conditions by observing the changes in beating heart muscle cells in culture. In particular, the effect of ouabain on contraction velocity of chicken heart muscle cells in culture was observed by electrically stimulating these cells.

The heart muscle cells to be stimulated and observed were cultured in a 25 cm² plastic culture flask using a growth medium modified by the addition of potassium and calcium electrolytes. A rubber stopper was inserted in the neck of the flask; the stopper was perforated by two steel electrodes for external cell stimulation, and by two syringe needles, one for delivering the ouabain into the chamber (long needle) and one for aspiration of the medium (short needle) . The electrodes and the needles were bent so that the electrodes were placed in the medium just above the cell monolayer, and the tips of both needles dipped into the medium. The cells were electrically driven by the steel electrodes connected to a Grass SD9 stimulator (Grass Instruments, Quincy, MA) with a pacing rate of 90 - 150 pulses/min, at 10 - 100 V, and a pulse duration of about 5 msec.

Using this device, pulsation of single cells could be continuously monitored under stable conditions over an observation period of about 60 minutes. After a period of 60 - 90 minutes, the heart cells would stop beating. Inspection of the device at the conclusion of an experiment periodically revealed burn marks on the chamber itself, near the electrodes.

When this device was used for longer periods, e.g., three hours, and with a larger chamber which required higher voltage, burning in the chamber occurred every time. Use of this device for the longer periods would be impossible not only because of the burning, but also due to corrosion of the electrodes, as well as contamination. Therefore, such a device is not suitable for biochemical and molecular biological assays of heart muscle cells which require stimulation ^■A. of a large number of cells over long periods of time

(days or weeks) .

_* 5 Other attempts to develop a device for stimulation of heart muscle cells include a round chamber with round electrodes. The results achieved with this device were not satisfactory, as only portions of the cells in the chamber would beat. This

10 lack of uniformity in the beating of the cells was due to the inhomogeneity of the electrical stimulation field as a result of the round design.

Accordingly, prior to the development of the present invention, no single device was capable of

15 uniformly stimulating a large number of heart muscle cells, so that they will contract or beat, over long periods of time as required for biochemical and molecular biological assays of heart muscle cells. It is therefore an object of the present invention to

20 provide a method and apparatus suitable for continuous uniform stimulation of a large number of heart muscle cells, so that they will contract or beat, over long periods of time. It is a further object of this invention to provide a method and apparatus suitable

25 for continuous uniform stimulation of heart muscle cells, so that they will contract or beat, for biochemical and molecular biological assays of these cells. It is an advantage of the present invention that the stimulation parameters, such as frequency,

30 pulse duration, and voltage, can be easily changed.

SUMMARY OF THE INVENTION

---*

The present invention is a device which is

35 capable of uniformly stimulating a large number of heart muscle cells, so that they will contract or beat, over long periods of time as required for biochemical and molecular biological assays of heart muscle cells. The device includes a chamber in which the cells are cultured and a chamber insert which contains the electrodes for generation of the electrical stimulation field. The chamber insert may be sufficiently heat resistant so that it can be sterilized in an autoclave. The electrodes can be made of graphite, platinum, or any similar material of sufficiently low resistance to prevent burning in the chamber when the system is operated for a period of at least 6 hours. The electrodes are configured so as to produce a homogeneous electric field to uniformly stimulate the cells in the chamber. The homogeneous field can be achieved through use of a pair of parallel electrodes.

In one aspect of the invention, parallel electrodes are used which are at least 16 cm in length; these electrodes may be made of graphite. In another aspect of the invention, a chamber with an area of at least 75 cm² is used. To produce the electric field, the electrodes are connected to an amplifier, which produces a signal with alternating polarity. The amplifier can be connected to the electrodes by wire made of platinum, nickel and chromium, or any similar material with low resistance and low corrosion. The amplifier can produce a range of outputs based upon the stimulation parameters it receives. . The stimulation parameters include the frequency or rate at which the pulse is generated, the voltage of the generated pulse, and the duration of the generated pulse. In one aspect of the invention, a frequency range of 0.5 - 6 Hz (30 pulses/min - 360 pulses/min) is used with a voltage range of 50 - 150 V at a pulse duration of approximately 5 msec for continuous stimulation for a period of 5 days.

Furthermore, the present invention includes a method for stimulating heart muscle cells which includes the steps of: culturing the heart muscles cells in the chamber; introducing the chamber insert

^'* with the electrodes into the chamber; producing a homogenous electric field to provide uniform

*5 stimulation of the cells in the chamber; and stimulating the cells with the electric field for a period of at least 6 hours. An additional step of sterilizing the chamber insert in an autoclave can be done prior to introducing the chamber insert into the

10 chamber. The electrodes for this method can be made of graphite or platinum, and can be connected to an amplifier which produces a signal of alternating polarity. The connection between the electrodes and the amplifier can be a wire made of platinum or nickel

15 and chromium. The electric field for this method can be continuously produced at a rate of 2 Hz using a 5 msec pulse duration for a period of 5 days.

BRIEF DESCRIPTION OF THE DRAWINGS

20

Various objects, features, and advantages of the present invention will be more fully appreciated as the same becomes better understood from the following detailed description of the present invention when 25 considered in connection with the accompanying drawings, in which:

FIGURE 1 is a perspective view of the bottom chamber and chamber insert;

FIGURE 2 is a block diagram showing the complete 30 stimulation system; and

FIGURE 3 is a schematic of the amplifier shown in FIGURE 2.

DETAILED DESCRIPTION OF THE DRAWINGS

35

With continuing reference to the drawing figures in which similar reference numerals are used throughout the description to describe similar features of the invention, the bottom chamber 200 is shown in FIGURE 1. In the preferred embodiment, a plastic chamber having an area of 175 cm² is used, such as is commercially available from Nunclon

Plastics (Roskilde, Denmark) . The size of the chamber used with this device may be varied. However, since it usually will be desired to stimulate a large number of cells, the chambers will normally have an area of at least 75 cm^2- The heart muscle cells are cultured in the chamber; neonatal heart cells attach easily to the chamber without the addition of any special substance. However, when adult heart cells are used, such as adult rat ventricular myocytes (ARVM) , the protein laminin is added to the chamber which provides a substance to which the adult cells attach. A medium is then added to the chamber. The medium contains the constituents required to support cell metabolism, such as electrolytes, amino acids, and glucose. The medium and cells within the chamber are indicated generally at 220. The medium is changed every 24 hours by opening the connector 210 at the neck of the chamber in a sterile environment. The medium is then aspirated from the chamber and replaced with fresh medium.

The chamber insert 100 is illustrated in FIGURE 1. The insert cover 110 is made of heat resistant LEXAN™ plastic so that it may be sterilized in an autoclave. This prevents infection in the long-term culture. The insert cover 110 has a cut out 160 for the connector 210 of the bottom chamber. Two parallel electrodes 130 are attached to the insert cover by the electrode support rods 120. The electrodes are parallel in order to generate a sufficiently homogeneous electric field to provide substantially uniform stimulation of the cells in the chamber. In the preferred embodiment, these electrodes are composed of pure graphite and are cylindrical in shape, with a length of 16.5 cm and a diameter of 0.65 cm. These electrodes are much larger than those used in the Werdan and Erdmann device in order to decrease the resistance generated. When too much resistance is generated, the result is burning in the chamber.

The choice of material for the electrodes will also affect the resistance. Additionally, the electrode material can affect cell performance and can degrade the long-term use of the device because of corrosion. Graphite is one material with low resistance and low corrosion that will not adversely affect cell performance. Platinum is another such material. Pure nickel-chromium is not a suitable material for the electrodes because it interferes with cell performance.

The electrodes are driven by an external amplifier (shown in FIGURE 2 as 320) . The electrodes are connected to the amplifier by the connector 150 and the wire 140. The connector 150 can be a standard commercial connector, such as a "banana" plug. Similar constraints apply to the choice of material for the wire as for the electrode. That is, the wire should have low resistance and low corrosion. Although the wire 140 is not in the medium itself, the environment is humid, so a material very resistant to corrosion for long periods is preferred, such as platinum. Nickel-chromium can also be used for the wire, as the wire is not in contact with the medium so it will not adversely affect cell performance.

The complete system 300 for long term stimulation of heart muscle cells is illustrated in FIGURE 2. The chamber insert is shown inside the bottom chamber 200. The electrodes 130 are connected to the amplifier 320 by the wires 140, through the connectors 150, to the cable 340. The amplifier generates an output signal of alternating polarity which is transmitted to the electrodes by the cable 340 and wires 140. The amplifier 320 is connected to a computer 310 by the cable 330. In the preferred embodiment, the computer 310 is an IBM PC, or compatible device, with an ADA 2100 (Real-Time Devices) board installed which outputs an electric signal to the amplifier 320 (± 2.5 V). The amplifier is composed of off-the-shelf components, and amplifies the signal received from the computer in order to provide sufficient voltage levels to the electrodes 130 for the stimulation of the heart muscle cells. Through use of the computer, the stimulation parameters can be easily regulated or changed. The stimulation parameters include the frequency or rate at which the pulse is generated, the voltage of the generated pulse, and the duration of the generated pulse.

A schematic of the amplifier 320 is shown in FIGURE 3. Input from the computer is received at input point 332, and the amplified signal is output to the electrodes at output point 342. A 115 V source is input at 322, with a fuse switch 324, and connected to two transformers, TR1 and TR2. In the preferred embodiment, Stancor P-6411 transformers are used for both TR1 and TR2. Preferably, diodes Dl - D4 are DFR604 diodes, and Cl and C2 are Mallory 1200 MFD/250 V capacitors. PI is a 10 turn potentiometer at 10 K ohm. The preferred value of Rl is 10 K ohm, of R2 is 1 K ohm, of R3 is 100 K ohm, of R4 is 2.7 ohm, and of C3 is 3.3 picofarad. Ul is preferably an Apex PA85 with heat sinks. The amplifier can generate signals up to approximately 150 V. However, the value of the components can be varied by one of ordinary skill in the art to increase the maximum output voltage of the amplifier so that more than one chamber can be connected to a single amplifier.

Commercial stimulators, such as the Grass stimulator, could be connected directly to the electrodes of the present invention. However, this is not the preferred mode as these stimulators typically have a low level DC leakage current between pulses. This leakage current causes continuous electrolysis

^■>5 and the generation of oxygen and hydrogen. This then continually changes the pH of the medium so that the medium would have to be continuously changed to provide an environment with stable pH for the cells over the entire duration of the test. Additionally, 0 these stimulators typically do not have sufficient output voltage to drive larger chambers or more than one chamber. However, a commercial stimulator, such as the Grass stimulator, could be connected to the amplifier of the present invention, thus providing an 5 input signal to the amplifier. The amplifier would then amplify the Grass stimulator signal to provide the higher drive power required for use with larger chambers or for use with more than one chamber.

This system has been used with numerous standard 0 disposable 175 cm² chambers containing cultures of adult rat ventricular myocytes (ARVM) ; each chamber contained approximately 3-4 x 10⁶ cardiac myocytes. Calcium-tolerant myocytes were enzymatically isolated using a Langendorff technique as described by Claycomb 5 and Palazzo (Dev. Biol. 80, 466-482 (1980)) with several modifications. The final cell suspension was layered twice over a 6% BSA gradient to minimize the number of non-myocyte cells. Myocytes were cultured on laminin in serum-free, defined medium M199 that was 0 supplemented with albumin (2 mg/ml BSA) , L-carnitine (2 mM) , taurine (5 mM) , insulin (0.1 microM) , triiodothyronine (T₃) (0.1 nM) , pyruvate (2.5 mM) , and

* penicillin (100 units/ml) /streptomycin (100 microg/ml) . 5 With parallel 16.5 cm graphite electrodes, up to

4 x 10⁶ ARVM per chamber could be stimulated uniformly at pulse frequencies from 0.5 - 6 Hz (30 pulses/min - 360 pulses/min) - A number of chambers have been stimulated concurrently for 5 days at 2 Hz with a pulse duration of approximately 5 msec, with 80% of the ARVM beating per chamber. Additionally, chambers have been uniformly stimulated at 3 Hz with a pulse duration of approximately 5 msec for 7 days. This system can, of course, be operated for shorter periods of time. The advantages of this device, i.e., its capability to be sterilized and to continuously operate without burning or corrosive effects, can be seen in a period as short as six hours. As this system can provide continuous uniform stimulation of a large number of cells, so that they will contract or beat, for long periods of time, it will allow for biochemical and molecular biological assays of the cells. Additionally, it will permit the investigation of the interaction between cells in the presence of active beating heart muscle in a co-culture system.

The invention which is intended to be protected herein should not be construed as limited to the particular forms disclosed, as these are to be regarded as illustrative rather than restrictive. For example, the electrodes can be configured in any way that will produce a homogeneous electrical stimulation field. The electrodes may be made of any material which has low resistance, low corrosion, and does not adversely affect cell performance. By changing the stimulation parameters, the device of the present invention could be used for long term stimulation of other muscle cells. The device could also be used to study the damaging effect on the cells by stimulating them with much higher voltages.

Variations and changes may be made by those skilled in the art without departing from the spirit of the invention. Accordingly, the foregoing detailed description should be considered exemplary in nature and not limited to the scope and spirit of the invention as set forth in the following claims.

Claims

WHAT IS CLAIMED IS:

1. Apparatus for stimulation of heart muscle cells for long periods of time, comprising: a chamber; a chamber insert; and means for stimulating comprising electrodes, wherein said electrodes produce an electric field sufficiently homogeneous to provide substantially uniform stimulation of said cells within said chamber, and the resistance generated by said electrodes is sufficiently low to prevent burning in said chamber while stimulating said cells for a period of at least 6 hours.

2. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 1, wherein said electrodes comprise graphite.

3. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 1, wherein said electrodes comprise platinum.

4. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 1, wherein said means for stimulating further comprises an amplifier, wherein said amplifier is connected to said electrodes by a wire, and said amplifier is capable of producing an output signal of alternating polarity.

5. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 4, wherein said output signal of said amplifier is carried by said wire, said wire comprising platinum.

6. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 4, wherein said output signal of said amplifier is carried by said wire, said wire comprising nickel and chromium.

7. Apparatus for stimulation of heart muscle cells < 5 for long periods of time according to claim 1, wherein said means for stimulating is capable of providing continuous stimulation at a rate of 2 Hz with a pulse duration of 5 msec for a period of 5 days.

10 8. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 4, wherein said output signal is in the range of 50 - 150 volts.

9. Apparatus for stimulation of heart muscle cells 15 for long periods of time according to claim 8, wherein said output signal has a duration of 5 msec at a rate in the range of 0.5 - 6 Hz.

10. Apparatus for stimulation of heart muscle cells 20 for long periods of time according to claim 1 or 4, wherein the area of said chamber is at least 75 cm².

11. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 1 or 4,

25 wherein said electrodes are a pair of parallel electrodes wherein each said electrode is at least 16 cm in length.

12. Apparatus for stimulation of heart muscle cells 30 for long periods of time according to claim 10, wherein said electrodes comprise graphite.

_'"

13. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 10,

35 wherein said means for stimulating is capable of providing continuous stimulation for a period of 5 days.

14. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 12, wherein said means for stimulating is capable of providing continuous stimulation for a period of 5 days.

15. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 11, wherein said electrodes comprise graphite.

16. Apparatus for stimulation of heart muscle cells for long periods of time according to claim 11, wherein said means for stimulating is capable of providing continuous stimulation for a period of 5 days.

17. Apparatus for stimulation of heart muscle cells for long periods of time, comprising: a chamber; a chamber insert comprising graphite electrodes wherein said chamber insert is sufficiently heat resistant to enable it to be sterilized by an autoclave; and an amplifier connected to said electrodes such that said electrodes produce an electric field sufficiently homogeneous to provide substantially uniform stimulation of said cells within said chamber while stimulating said cells for a period of at least 6 hours.

18. Method for stimulating heart muscle cells comprising the steps of: a) culturing said heart muscle cells in a chamber; b) introducing a chamber insert into said chamber wherein said chamber insert comprises electrodes; c) producing an electric field from said electrodes such that said electric field is sufficiently homogeneous to provide substantially uniform stimulation of said cells within said

« 5 chamber; and d) stimulating said cells with said electric field for a period of at least 6 hours.

19. Method for stimulating heart muscle cells 10 according to claim 18, further comprising the step of sterilizing said chamber insert by using an autoclave prior to the step of introducing said chamber insert into said chamber.

15 20. Method for stimulating heart muscle cells according to claim 18, wherein said electrodes comprise graphite.

21. Method for stimulating heart muscle cells 20 according to claim 18, wherein said electrodes comprise platinum.

22. Method for stimulating heart muscle cells according to claim 18, wherein said electrodes are

25 connected to an amplifier by a wire, and said amplifier is capable of producing an output signal of alternating polarity.

23. Method for stimulating heart muscle cells 30 according to claim 22, wherein said output signal of * said amplifier is carried by said wire, said wire comprising platinum.

24. Method for stimulating heart muscle cells 35 according to claim 22, wherein said output signal of said amplifier is carried by said wire, said wire comprising nickel and chromium.

25. Method for stimulating heart muscle cells according to claim 19, wherein said electric field is continuously produced at a rate of 2 Hz with a 5 msec duration over a period of 5 days.